激活突变引发SHP2蛋白相分离对小鼠间充质干细胞增殖能力的影响及其机制

Effect and mechanism of SHP2 protein phase isolation induced by activated mutation on proliferation of mouse mesenchymal stem cells

目的构建SHP2 E76K突变的小鼠模型,利用其间充质干细胞(MSC)研究激活突变引发的SHP2蛋白相分离对其细胞增殖能力的影响及其机制。方法将Ptpn11 E76K-neo/+的C57BL/6J小鼠与Mx1-cre工具鼠杂交,得到所需要的Mx1-cre;Ptpn11^(+/+)和Mx1-cre;Ptpn11^(E76K/+)小鼠,并对后者注射pI-pC以诱导Cre酶的表达,使Ptpn11在骨髓MSC中突变。从Mx1-cre;Ptpn11^(+/+)和Mx1-cre;Ptpn11^(E76K/+)小鼠体内分离培养MSC,通过细胞免疫荧光染色确定所分离的细胞为MSC。以Ptpn11^(+/+)的MSC为对照组,Ptpn11^(E76K/+)的MSC为实验组,通过加药将两种细胞分成6组:Ptpn11^(+/+)组;Ptpn11^(+/+)+SHP099组;Ptpn11^(+/+)+ET070组;Ptpn11^(E76K/+)组;Ptpn11^(E76K/+)+SHP099组;Ptpn11^(E76K/+)+ET070组。免疫荧光染色法观察6组细胞内SHP2蛋白相分离的差异,Western blot法检测6组MSC中SHP2蛋白表达水平的差异。CCK-8检测相分离被影响之后细胞增殖能力的改变。提取实验组与对照组细胞总蛋白通过Western blot法检测ERK/p-ERK、AMPK/p-AMPK、mTOR/p-mTOR等蛋白的表达水平。结果小鼠基因型鉴定证实得到Mx1-cre;Ptpn11^(E76K/+)和Mx1-cre;Ptpn11^(+/+)小鼠并分离出原代Ptpn11^(+/+)和Ptpn11^(E76K/+)骨髓MSC。与Ptpn11^(+/+)组相比,Ptpn11^(E76K/+)组的SHP2蛋白产生更多的相分离冷凝物;与Ptpn11^(E76K/+)组相比,Ptpn11^(E76K/+)+SHP099组和Ptpn11^(E76K/+)+ET070组SHP2蛋白凝聚成的冷凝物在均明显减少;Western blot检测各组SHP2蛋白表达水平差异无统计学意义;与Ptpn11^(+/+)组相比,Ptpn11^(+/+)+SHP099组和Ptpn11^(+/+)+ET070组MSC的增殖能力下降,细胞内p-ERK和p-mTOR蛋白表达减少,p-AMPK蛋白表达增加;与Ptpn11^(E76K/+)组相比,Ptpn11^(E76K/+)+SHP099组和Ptpn11^(E76K/+)+ET070组MSC的增殖能力下降,细胞内p-ERK和p-mTOR蛋白表达减少,p-AMPK蛋白表达增加。结论SHP2的相分离通过刺激AMPK-mTOR信号通路的表达参与了SHP2 E76K激活突变的MSC的增殖能力改变。

Objective To investigate the effect of SHP2 protein phase separation induced by activation mutation on cell proliferation and its mechanism through construct a mouse model of SHP2 E76K mutation.Methods Hybrid PTPN11 E76K-NEO/+C57BL/6J mouse were hybridizedwith Mx1-cre tool mice to obtain the required Mx1-cre;Ptpn11^(+/+)and Mx1-cre;Ptpn11^(E76K/+).The later genotype mice were injected with pI-pC to induce the expression of Cre enzyme and mutate Ptpn11E76K in bone marrow mesenchymal stem cells(MSC).The Mx1-cre;Ptpn11^(+/+)and Mx1-cre;Ptpn11^(E76K/+)genotype mice′s cells were isolated and cultured in vitro and identified as MSCs by immunofluorescence staining.With Ptpn11^(+/+)MSC as the control group and Ptpn11^(E76K/+)MSC as the experimental group,the two kinds of cells were divided into 6 groups by adding drugs:Ptpn11^(+/+)group;Ptpn11^(+/+)+SHP099 group;Ptpn11^(+/+)+ET070 group;Ptpn11^(E76K/+)group;Ptpn11^(E76K/+)+SHP099 group;Ptpn11^(E76K/+)+ET070 group.The differences of SHP2 protein phase separation in the six groups were observed by immunofluorescencestaining,and the differences of SHP2 protein expression were detectedby Western blot.CCK-8 was used to observe the changes of cell proliferation after phase separation was affected.Western blot was used to detect the expression levels of ERK/p-ERK,AMPK/p-AMPK,mTOR/p-mTOR and other molecules between the six groups.Results Genotypes Mx1-cre;Ptpn11^(E76K/+)and Mx1-cre;Ptpn11^(+/+)mice were obtained by genotyping,and the primary MSCs were isolated.Compared with Ptpn11^(+/+)group,SHP2 proteins in the Ptpn11^(E76K/+)group produced more phase separation condensates,and compared with Ptpn11^(E76K/+)group,the SHP2 proteins in the Ptpn11^(E76K/+)+SHP099 and Ptpn11^(E76K/+)+ET070 groups produced less phase separation condensates.No difference in SHP2 protein expression levels between groups was detected by Western blot.Compared with Ptpn11^(+/+)group,the proliferation ability of MSC in Ptpn11^(+/+)+SHP099 and Ptpn11^(+/+)+ET070 groups decreased,the expression of p-ERK and p-mTOR decreased,and the expression of p-AMPK protein increased.Compared with Ptpn11^(E76K/+)group,the proliferation ability of MSC in Ptpn11^(E76K/+)+SHP099 and Ptpn11^(E76K/+)+ET070 groups decreased,the expression of p-ERK and p-mTOR decreased,and the expression of p-AMPK protein increased.Conclusion SHP2 phase isolation is involved in the alteration of proliferative capacity of SHP2 E76K-activated MSCs by stimulating the expression of AMPK-mTOR signaling pathway.